Drug-induced liver toxicity represents an important healthcare issue because it causes significant morbidity and mortality and can be extremely difficult to predict. Elucidating the mechanism(s) of drug-induced liver toxicity, therefore, is essential for the design of safer therapeutic agents. The antipyretic and analgesic drug, acetaminophen (APAP), has been the most widely studied of all drugs with hepatotoxic potential. It continues to be a significant cause of liver injury and lethality in humans. Studies have suggested that APAP-induced liver injury may be related to protein arylation, oxidative stress, disruption of calcium and mitochondrial homeostasis, alteration of transcription pathways, and the induction of cell death pathways. However, the precise molecular events occurring within the liver following APAP insult have still not been deciphered, and only a limited number of pathways involved in controlling toxicity progression have been identified. Using the NIEHS cDNA microarray technique, we conducted an in vivo gene expression profiling approach for studying the mechanism(s) of APAP-induced hepatotoxicity. We have evaluated mRNA from target tissues (liver and kidney) as well as from blood in order to ascertain the usefulness of blood to serve as a biomarker for tissue-specific toxicity. Many of the genes have not previously been reported and/or thoroughly investigated in the context of drug toxicity and, therefore, represent novel factors for further study of the mechanism(s) of APAP-induced liver injury. Similar factors may also play a role in the hepatotoxic potential of other drugs or environmental agents we will study in the future.